Proteomics

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A universal method for the rapid isolation of all known classes of functional small RNAs

ABSTRACT: Diverse classes of regulatory small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Based the conserved biochemical properties intrinsic to all ARGONAUTEs, we have developed a universal, 15-min benchtop extraction procedure allowing simultaneous purification of all classes of RISC-associated sRNAs, without prior knowledge of the sample’s -intrinsic ARGONAUTE repertoires. Optimized as a user-friendly kit, the method –coined “TraPR” for Trans-kingdom, rapid, affordable Purification of RISCs– operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct sRNA deep-sequencing, with TraPR-generated libraries being qualitatively at least on-par with those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel excisions. TraPR considerably improves the quality and consistency of sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots and mammalian plasma, and regardless of RNA contaminants or samples’ RNA-degradation status.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Caenorhabditis Elegans

SUBMITTER: Julia Svozil  

LAB HEAD: Olivier Voinnet

PROVIDER: PXD015395 | Pride | 2021-09-08

REPOSITORIES: Pride

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20180806_009_S183087_CE_I2_2_Group_2.raw Raw
20180806_010_S183088_CE_E2_2_Group_2.raw Raw
20180806_012_S183089_CE_HS2_2_Group_2.raw Raw
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Publications

A universal method for the rapid isolation of all known classes of functional silencing small RNAs.

Grentzinger Thomas T   Oberlin Stefan S   Schott Gregory G   Handler Dominik D   Svozil Julia J   Barragan-Borrero Veronica V   Humbert Adeline A   Duharcourt Sandra S   Brennecke Julius J   Voinnet Olivier O  

Nucleic acids research 20200801 14

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the sa  ...[more]

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